Defects in apoptosis signaling pathway and upregulation of apoptosis inhibitory factors confer resistance of human cancer cells to therapeutic agents. A promising strategy to overcome the resistance is to target apoptosis machinery directly. Increasing evidence indicates that inhibitor-of-apoptosis proteins (IAPs) are upregulated in human tumor cells. We found that expression of IAP counteracting protein genes, such as dominant negative survivinT34A mutant, XIAP associated factor 1 (XAF1) and active Smac, induce apoptotic cell death preferentially in tumor cell lines but have no apparent effect on normal cell lines. To determine the molecular bases of the differential apoptotic response in tumor and normal cell lines, we examined the levels of apoptotic and anti-apoptotic factors in human cancer and normal cell lines. We found that many human cancer cell lines have constitutively activated caspase activities and yet are not undergoing apoptosis. We also detected high levels of IAPs, such as survivin and XIAP, in the tumor cell lines. Based on these observations, we hypothesize that the presence of constitutively activated caspases and upregulated IAPs in human tumor cells, but not in normal cells, confers selectivity in induction of apoptotic cell death in tumor cells by expression of survivinT34A, XAF1 and/or active Smac genes. A combination of downregulation of IAP function with chemotherapy agents further enhances anti-tumor effects in tumor cell lines as well as in human tumor xenograft models. To test the hypothesis, we will determine whether constitutively activated caspases are required for selective induction of apoptosis in tumor cell lines by expression of LAP counteracting protein genes in the presence of caspase inhibitors or with co-expression of an autocatalytic Rev-caspase 3 gene. We will further determine whether apoptosis can be induced in normal cell lines by expressing IAP counteracting protein genes when the cells are engineered to express high levels of both activated caspase 3 and XIAP. Since human tumor cells express high levels of survivin and XIAP, it is necessary to inhibit the function of both proteins to release the blockage of IAPs on the apoptotic pathway. In the proposed study, we will determine the most effective combination of lAP counteracting protein genes to achieve maximum apoptosis induction in human tumor cells. Finally, we will determine whether a combination of expression of IAP counteracting protein genes with chemotherapy drug docetaxel or HSV-TK/GCV suicidal gene therapy increases the anti-tumor effect in human breast and pancreatic cancer cell lines as well as in human tumor xenograft models. Results from this research should allow us to determine if IAPs are novel molecular targets for the development of cancer-specific therapeutic approaches.